Some membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins. Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol (GPI) groups. Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction.
Ras genes are members of the Ras supergene family of genes, present in various species, including humans and rodents. This supergene family consists of 50 or more genes that encode structurally related proteins which possess a guanine triphosphate binding site and intrinsic guanine triphosphatase (GTPase) activity. Many members of this superfamily of proteins are critical for diverse intracellular signaling pathways. The Ras subfamily in humans of this supergene family includes 36 genes that encode 39 proteins (Wennerberg et al., “The Ras Superfamily at a Glance,” J. Cell Science 118(5):843-846 (2005)). Among these are the H-Ras, K-Ras (also known as Kras2), N-Ras, Rap, Rheb and Ral genes. The chromosomal locations of each of the human Ras genes is known in the art.
Ras proteins are attractive targets for anti-cancer drugs because they are believed to be involved in at least 30% of human cancers. Ras proteins must associate with cellular membranes in order to function and, therefore, one strategy for developing anti-Ras drugs is to inhibit membrane targeting of the Ras proteins. Central to the mechanism whereby three of the four iso forms of Ras associate with membranes is their post-translational modification with a farnesyl lipid and, in the case of N-Ras and H-Ras, one or two palmitate lipids.
Although mammalian genomes contain three Ras genes, mutations in K-Ras are most frequently associated with human cancer (Bos, “Ras Oncogenes in Human Cancer: A Review,” Cancer Res. 49:4682-4689 (1989)). Therefore, properties that are specific to K-Ras are of particular significance to cancer biologists since they might be exploited in the development of anti-cancer drugs. The differential biology of Ras iso forms is generated, in large part, by distinct membrane targeting sequences. Membrane association of all Ras isoforms requires prenylation (i.e., farnesylation), proteolysis, and carboxyl methylation of a C-terminal CAAX (SEQ ID NO:1) motif (where C stands for cysteine, A for an aliphatic amino acid and X for any amino acid). Plasma membrane targeting of the principal splice variant of K-Ras also requires a unique polybasic region adjacent to the CAAX motif (Hancock et al., “A Polybasic Domain or Palmitoylation is Required in Addition to the CAAX Motif to Localize p21ras to the Plasma Membrane,” Cell 63:133-139 (1990); Jackson et al., “Polylysine Domain of K-Ras 4B Protein is Crucial for Malignant Transformation,” Proc. Natl. Acad. Sci. USA 91:12730-12734 (1994); Choy et al., “Endomembrane Trafficking of Ras: The CAAX Motif Targets Proteins to the ER and Golgi,” Cell 98:69-80 (1999)).
K-Ras thus falls into a broad class of proteins that are anchored to the cytoplasmic face of the plasma membrane by virtue of post-translational modification with lipids that act in conjunction with polybasic stretches of polypeptide. Whereas the lipid moieties are thought to insert into the phospholipid bilayer, the polybasic regions are believed to associate with the anionic head groups of inner leaflet phospho lipids (Leventis et al., “Lipid-Binding Characteristics of the Polybasic Carboxy-Terminal Sequence of K-Ras4B,” Biochemistry 37:7640-7648 (1998)). Considering the significance of this class of proteins, there is a need for information about the mechanism of cellular membrane interaction and control of such interaction.
The present invention is directed to overcoming these and other deficiencies in the art.